The present invention is directed to nucleic acid and amino acid sequences and constructs, and methods related thereto.
Isoprenoids are ubiquitous compounds found in all living organisms. Plants synthesize a diverse array of greater than 22,000 isoprenoids (Connolly and Hill (1992) Dictionary of Terpenoids, Chapman and Hall, New York, N.Y.). In plants, isoprenoids play essential roles in particular cell functions such as production of sterols, contributing to eukaryotic membrane architecture, acyclic polyprenoids found in the side chain of ubiquinone and plastoquinone, growth regulators like abscisic acid, gibberellins, brassinosteroids or the photosynthetic pigments chlorophylls and carotenoids. Although the physiological role of other plant isoprenoids is less evident, like that of the vast array of secondary metabolites, some are known to play key roles mediating the adaptative responses to different environmental challenges. In spite of the remarkable diversity of structure and function, all isoprenoids originate from a single metabolic precursor, isopentenyl diphosphate (IPP) (Wright, (1961) Annu. Rev. Biochem. 20: 525-548; and Spurgeon and Porter, (1981) in Biosynthesis of Isoprenoid Compounds., Porter and Spurgeon eds (John Wiley, New York) Vol. 1, pp1-46).
A number of unique and interconnected biochemical pathways derived from the isoprenoid pathway leading to secondary metabolites, including tocopherols, exist in chloroplasts of higher plants. Tocopherols not only perform vital functions in plants, but are also important from mammalian nutritional perspectives. In plastids, tocopherols account for up to 40% of the total quinone pool.
Tocopherols and tocotrienols (unsaturated tocopherol derivatives) are well known antioxidants, and play an important role in protecting cells from free radical damage, and in the prevention of many diseases, including cardiac disease, cancer, cataracts, retinopathy, Alzheimer""s disease, andneurodegeneration, and have been shown to have beneficial effects on symptoms of arthritis, and in anti-aging. .Vitamin E is used in chicken feed for improving the shelf life, appearance, flavor, and oxidative stability of meat, and to transfer tocols from feed to eggs. Vitamin E has been shown to be essential for normal reproduction, improves overall performance, and enhances immunocompetence in livestock animals. Vitamin E supplement in animal feed also imparts oxidative stability to milk products.
The demand for natural tocopherols as supplements has been steadily growing at a rate of 10-20% for the past three years. At present, the demand exceeds the supply for natural tocopherols, which are known to be more biopotent than racemic mixtures of synthetically produced tocopherols. Naturally occurring tocopherols are all d-stereomers, whereas synthetic xcex1-tocopherol is a mixture of eight d,l-xcex1-tocopherol isomers, only one of which (12.5%) is identical to the natural d-xcex1-tocopherol. Natural d-xcex1-tocopherol has the highest vitamin E activity (1.49 IU/mg) when compared to other natural tocopherols or tocotrienols. The synthetic xcex1-tocopherol has a vitamin E activity of 1.1 IU/mg. In 1995, the worldwide market for raw refined tocopherols was $1020 million; synthetic materials comprised 85-88% of the market, the remaining 12-15% being natural materials. The best sources of natural tocopherols and tocotrienols are vegetable oils and grain products. Currently, most of the natural Vitamin E is produced from xcex3-tocopherol derived from soy oil processing, which is subsequently converted to xcex1-tocopherol by chemical modification (xcex1-tocopherol exhibits the greatest biological activity).
Methods of enhancing the levels of tocopherols and tocotrienols in plants, especially levels of the more desirable compounds that can be used directly, without chemical modification, would be useful to the art as such molecules exhibit better functionality and biovailability.
In addition, methods for the increased production of other isoprenoid derived compounds in a host plant cell is desirable. Furthermore, methods for the production of particular isoprenoid compounds in a host plant cell is also needed.
The present invention is directed to prenyltransferase (PT), and in particular to PT polynucleotides and polypeptides. The polynucleotides and polypeptides of the present invention include those derived from prokaryotic and eukaryotic sources.
Thus, one aspect of the present invention relates to isolated polynucleotide sequences encoding prenyltransferase proteins. In particular, isolated nucleic acid sequences encoding PT proteins from bacterial and plant sources are provided.
Another aspect of the present invention relates to oligonucleotides which include partial or complete PT encoding sequences.
It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for transcription or transcription and translation (expression) of prenyltransferase. In particular, constructs are provided which are capable of transcription or transcription and translation in host cells.
In another aspect of the present invention, methods are provided for production of prenyltransferase in a host cell or progeny thereof. In particular, host cells are transformed or transfected with a DNA construct which can be used for transcription or transcription and translation of prenyltransferase. The recombinant cells which contain prenyltransferase are also part of the present invention.
In a further aspect, the present invention relates to methods of using polynucleotide and polypeptide sequences to modify the tocopherol content of host cells, particularly in host plant cells. Plant cells having such a modified tocopherol content are also contemplated herein.
The modified plants, seeds and oils obtained by the expression of the prenyltransferases are also considered part of the invention.